Opioid drugs have various effects on perception of pain, consciousness, motor control, mood, and autonomic function and can also induce physical dependence (Koob et al., 1992). The endogenous opioid system plays an important role in modulating endocrine, cardiovascular, respiratory, gastrointestinal and immune functions (Olson et al., 1989). Opioids exert their actions by binding to specific membrane-associated receptors located throughout the central and peripheral nervous system (Pert et al., 1973). The endogenous ligands of these opioid receptors have been identified as a family of more than 20 opioid peptides that derive from the three precursor proteins proopiomelanocortin, proenkephalin, and prodynorphin (Hughes et al., 1975; Akil, et al., 1984). Although the opioid peptides belong to a class of molecules distinct from the opioid alkaloids, they share common structural features including a positive charge juxtaposed with an aromatic ring that is required for interaction with the receptor (Bradbury et al., 1976).
Pharmacological studies have suggested that there are numerous classes of opioid receptors, including those designated .delta., .kappa., .mu. and .epsilon. (Simon, 1991; Lutz et al., 1992). The classes differ in their affinity for various opioid ligands and in their cellular distribution. The different classes of opioid receptors are believed to serve different physiological functions (Olson. et al., 1989; Simon, 1991; Lutz and Pfister, 1992). However, there is substantial overlap of function as well as of distribution. Biochemical characterization of opioid receptors from many groups reports a molecular mass of .apprxeq.60,000 Da for all three subtypes, suggesting that they could be related molecules (Loh et at., 1990). Moreover, the similarity between the three receptor subtypes is supported by the isolation of (i) anti-idiotypic monoclonal antibodies competing with both .mu. and .delta. ligands but not competing with .kappa. ligands (Gramsch et al., 1988; Coscia et al., 1991) and (ii) a monoclonal antibody raised against the purified .mu. receptor that interacts with both .mu. and .kappa. receptors (Bero et al., 1988).
Morphine interacts principally with .mu. receptors and peripheral administration of this opioid induces release of enkephalins (Bertolucci et al., 1992). The .delta. receptors bind with the greatest affinity to enkephalins and have a more discrete distribution in the brain than either .mu. or .kappa. receptors, with high concentrations in the basal ganglia and limbic regions. Thus, enkephalins may mediate part of the physiological response to morphine, presumably by interacting with .delta. receptors. Despite pharmacological and physiological heterogeneity, at least some types of opioid receptors inhibit adenylate cyclase, increase K.sup.+ conductance, and inactivate Ca.sup.2+ channels through a pertussis toxin-sensitive mechanism (Puttfarcken et al., 1988; Attali et al., 1989, Hsia et al., 1984). These results and others suggest that opioid receptors belong to the large family of cell surface receptors that signal through G proteins (Di Chiara et al., 1992; Loh et al., 1990).
The 6, 7 benzoinorphans such as ethylketocyclazocine label two populations of non-.mu., non-.delta. opioid binding sites in brain that are the .kappa. and .epsilon. sites (Chang et al., 1981). The benezencacctamide U-69, 593 has been shown to selectively label one of these two 6, 7 benzoinorphan sites which corresponds to the .kappa. opioid receptor site, but the other benzoinorphan site lacks a selective ligand (Nock et al., 1988). The nature and designation of the U-69, 593 insensitive benzoinorphan site has been debated, including suggestions that it might be a .kappa. opioid receptor subtype because of high affinity interactions with certain .kappa. opioid ligands. However dynorpyhin and other prodymorphin derived peptides presumed to be the endogenous ligands of the .kappa. opioid receptor had very low affinity for this site, which had high affinity for .beta. endorphin (Nock et al., 1993). This pharmacological selectivity profile corresponds to that of the epsilon (.epsilon.) opioid receptor, characterized as a dynorphin-insensitive, non-.mu., non-.delta. opioid binding site. The .epsilon. receptor was first hypothesized to exist based on bioassays involving the rat vas deferens and from radioligand binding studies in brain; however it has subsequently been shown to be the most abundant opioid binding site in brain (Nock et al., 1993).
Several attempts to clone cDNAs encoding opioid receptors have been reported. A cDNA encoding an opioid-binding protein (OBCAM) with .mu. selectivity was isolated (Schofield et al., 1989), but the predicted protein lacks transmembrane domains, presumed necessary for signal transduction. More recently, the isolation of another cDNA was reported, which was obtained by expression cloning (Xie et al., 1992). The deduced protein sequence displays seven putative transmembrane domains and is very similar to the human neuromedin K receptor. However, the affinity of opioid ligands for this receptor expressed in COS cells is two orders of magnitude below the expected value, and no subtype selectivity can be shown.
Many cell surface receptor/transmembrane systems consist of at least three membrane-bound polypeptide components: (a) a cell-surface receptor; (b) an effector, such as an ion channel or the enzyme adenylate cyclase; and (c) a guanine nucleotide-binding regulatory polypeptide or G protein, that is coupled to both the receptor and its effector.
G protein-coupled receptors mediate the actions of extracellular signals as diverse as light, odorants, peptide hormones and neurotransmitters. Such receptors have been identified in organisms as evolutionarily divergent as yeast and man. Nearly all G protein-coupled receptors bear sequence similarities with one another, and it is thought that all share a similar topological motif consisting of seven hydrophobic (and potentially .alpha.-helical) segments that span the lipid bilayer (Dohlman et al., 1987; Dohlman et al., 1991).
G proteins consist of three tightly associated subunits, .alpha., .beta. and .gamma. (1:1:1) in order of decreasing mass. Following agonist binding to the receptor, a conformational change is transmitted to the G protein, which causes the G.alpha.-subunit to exchange a bound GDP for GTP and to dissociate from the .beta..gamma.-subunits. The GTP-bound form of the .alpha.-subunit is typically the effector-modulating moiety. Signal amplification results from the ability of a single receptor to activate many G protein molecules, and from the stimulation by G.alpha.-GTP of many catalytic cycles of the effector.
The family of regulatory G proteins comprises a multiplicity of different .alpha.-subunits (greater than twenty in man), which associate with a smaller pool of .beta.- and .gamma.-subunits (greater than four each) (Strothman and Simon, 1991). Thus, it is anticipated that differences in the .alpha.-subunits probably distinguish the various G protein oligomers, although the targeting or function of the various .alpha.-subunits might also depend on the .beta..gamma. subunits with which they associate (Strothman and Simon, 1991).
Improvements in cell culture and in pharmacological methods, and more recently, use of molecular cloning and gene expression techniques have led to the identification and characterization of many seven-transmembrane segment receptors, including new sub-types and sub-sub-types of previously identified receptors. The .alpha..sub.1 and .alpha..sub.2 -adrenergic receptors once thought to each consist of single receptor species, are now known to each be encoded by at least three distinct genes (Kobilka et al., 1987; Regan et al., 1988; Cotecchia et al., 1988; Lomashey, 1990). In addition to rhodopsin in rod cells, which mediates vision in dim light, three highly similar cone pigments mediating color vision have been cloned (Nathans et al., 1986A, and Nathans et al., 1986B). All of the family of G protein-coupled receptors appear to be similar to other members of the family of G protein-coupled receptors (e.g., dopaminergic, muscarinic, serotonergic, tachykinin, etc.), and each appears to share the characteristic seven-transmembrane segment topography.
When comparing the seven-transmembrane segment receptors with one another, a discernible pattern of amine acid sequence conservation is observed. Transmembrane domains are often the most similar, whereas the amine and carboxyl terminal regions and the cytoplasmic loop connecting transmembrane segments V and VI can be quite divergent (Dohlman et al., 1987).
Interaction with cytoplasmic polypeptides, such as kinases and G proteins, was predicted to involve the hydrophobic loops connecting the transmembrane domains of the receptor. The challenge, however, has been to determine which features are preserved among the seven-transmembrane segment receptors because of conservation of function, and which divergent features represent structural adaptations to new functions. A number of strategies have been used to test these ideas, including the use of recombinant DNA and gene expression techniques for the construction of substitution and deletion mutants, as well as of hybrid or chimeric receptors (Dohlman et al., 1991).
With the growing number of receptor sub-types, G-protein subunits, and effectors, characterization of ligand binding and G protein recognition properties of these receptors is an important area for investigation. It has long been known that multiple receptors can couple to a single G protein and, as in the case of epinephrine binding to .beta..sub.2 - and .alpha..sub.2 -adrenergic receptors, a single ligand can bind to multiple functionally distinct receptor sub-types. Moreover, G proteins with similar receptor and effector coupling specificities have also been identified. For example, three species of human G.sub.i have been cloned (Itoh et al., 1988), and alternate mRNA splicing has been shown to result in multiple variants of G.sub.S (Kozasa et al., 1988). Cloning and over production of the muscarinic and .alpha..sub.2 -adrenergic receptors led to the demonstration that a single receptor sub-type, when expressed at high levels in the cell, will couple to more than one type of G protein.
Opioid receptors are known to be sensitive to reducing agents, and the occurrence of a disulfide bridge has been postulated as essential for ligand binding (Gioannini et al., 1989). For rhodopsin, muscarinic, and .beta.-adrenergic receptors, two conserved cysteine residues in each of the two first extracellular loops have been shown critical for stabilizing the functional protein structure and are presumed to do so by forming a disulfide bridge. Structure/function studies of opioid ligands have shown the importance of a protonated amine group for binding to the receptor with high affinity. The binding site of the receptor might, therefore, possess a critical negatively charged counterpart. Catecholamine receptors display in their sequence a conserved aspartate residue that has been shown necessary for binding the positively charged amine group of their ligands.
Given the complexity and apparent degeneracy of function of various opioid receptors, a question of fundamental importance is how, and under what circumstances do specific subtype and sub-sub-type receptors exert their physiological effect in the presence of the appropriate stimulatory ligand. A traditional approach to answering this question has been to reconstitute the purified receptor and G protein components in vitro. Unfortunately, purification schemes have been successful for only a very limited number of receptor sub-types and their cognate G-proteins. Alternatively, heterologous expression systems can be of more general usefulness in the characterization of cloned receptors and in elucidating receptor G protein coupling specificity (Marullo et al., 1988; Payette et al., 1990; King et al., 1990).
One such system was recently developed in yeast cells, in which the genes for a mammalian .beta..sub.2 -adrenergic receptor and G.sub.s .alpha.-subunit were coexpressed (King et al., 1990). Expression of the .beta..sub.2 -adrenergic receptor to levels several hundred-fold higher than in any human tissue was attained, and ligand binding was shown to be of the appropriate affinity, specificity, and stereoselectivity. Moreover, a .beta..sub.2 -adrenergic receptor-mediated activation of the pheromone signal transduction pathway was demonstrated by several criteria, including imposition of growth arrest, morphological changes, and induction of a pheromone-responsive promoter (FUS1) fused to the Escherichia coli lac Z gene (encoding .beta.-galactosidase) (King et al., 1990).
Finally, expression of a single receptor in the absence of other related sub-types is often impossible to achieve, even in isolated, non-recombinant mammalian cells. Thus, there has been considerable difficulty in applying the standard approaches of classical genetics or even the powerful techniques of molecular biology to the study of opioid receptors. In particular, means are needed for the identification of the DNA sequences encoding individual opioid receptors. Given such isolated, recombinant sequences, it is possible to address the heretofore intractable problems associated with design and testing of isoform-specific opioid receptor agonists and antagonists. The availability of cDNAS encoding the opioid receptors will permit detailed studies of signal-transduction mechanisms and reveal the anatomical distribution of the mRNAs of these receptors, providing information on their expression pattern in the nervous system. This information should ultimately allow better understanding of the opioid system in analgesia, and also the design of more specific therapeutic drugs.
Availability of polynucleotide sequences encoding opioid receptors, and the polypeptide sequences of the encoded receptors, will significantly increase the capability to design pharmaceutical compositions, such as analgesics, with enhanced specificity of function. In general, the availability of these polypeptide sequences will enable efficient screening of candidate compositions. The principle in operation through the screening process is straightforward: natural agonists and antagonists bind to cell-surface receptors and channels to produce physiological effects; certain other molecules bind to receptors and channels; therefore, certain other molecules may produce physiological effects and act as therapeutic pharmaceutical agents. Thus, the ability of candidate drugs to bind to opioid receptors can function as an extremely effective screening criterion for the selection of pharmaceutical compositions with a desired functional efficacy.
Prior methods tier screening candidate drug compositions based on their ability to preferentially bind to cell-surface receptors has been limited to tissue-based techniques. In these techniques, animal tissues rich in the receptor type of interest are extracted and prepared; candidate drugs are then allowed to interact with the prepared tissue and those found to bind to the receptors are selected tier further study. However, these tissue-based screening techniques suffer from several significant disadvantages. First, they are expensive because the source of receptor cell tissue--laboratory animals--is expensive. Second, extensive technical input is required to operate the screens. And, third, the screens may confuse the results because there are no tissues where only one receptor subtype is expressed exclusively. With traditional prior art screens you are basically looking at the wrong interactions or, at best, the proper interactions mixed in with a whole variety of unwanted interactions. An additional fundamental deficiency of animal tissue screens is that they contain animal receptors--ideal for the development of drugs for animals but of dubious value in human therapeutic agents.
The disadvantages of the prior art may be overcome by providing a polynucleotide transfected into suitable host cells which can express polypeptide sequences corresponding to opioid receptors, both in large quantities and through relatively simple laboratory procedures. The result is the availability of extremely specific receptor-drug interactions free from the competitive and unwanted interactions encountered in tissue-based screens. Further expression in a microorganism where no such endogenous receptors exist (e.g. yeast cells or mutant mammalian cell lines) can be useful for screening and evaluating sub-type-selective drugs (Marullo et al., 1988; Paycite et al., 1990; and King et al., 1990).